a) Brief study of basic metabolic pathways and formation of different secondary metabolites through these pathways- Shikimic acid pathway, Acetate pathways and Amino acid pathway. b) Study of utilization of radioactive isotopes in the investigation of Biogenetic studies.

1. Metabolic pathways
in higher plants
and their determination
DR. SIDDHI UPADHYAY
H.O.D. & ASSOCIATE PROFESSOR
Dept. of Pharmacognosy and Phytochemistry
SIGMA INSTITUTE OF PHARMACY

2. CONTENT
a) Brief study of basic metabolic pathways and
formation of different secondary metabolites through
these pathways- Shikimic acid pathway, Acetate
pathways and Amino acid pathway.
b) Study of utilization of radioactive isotopes in the
investigation of Biogenetic studies.

3. BASIC METABOLIC PATHWAYS

4. BASIC METABOLIC PATHWAYS
1. Calvin cycle
- precursor for carbohydrates
2. Acetate Mevalonate Pathway
- precursor for steroids, terpenoids, few alkaloids, few glycosides
3. Acetate Malonate Pathway
- precursor for fatty acids
4. Polyacetate Malonate Pathway
- precursor for anthraquinones, flavonoids
5. Shikimic Acid Pathway
- precursor for aromatic amino acids, tannins, flavonoids,
phenypropanoids, some alkaloids, some glycosides
6. Amino Acid Pathway
- precursor for aliphatic amino acids
7. Flavonoid Pathway
- precursor for flavonoids

5. S
H
I
K
I
M
I
C
A
C
I
D
P
A
T
H
W
A
Y

6. Acetate Mevalonate Pathway

7. Acetate
Malonate
Pathway

8. Amino Acid Pathways
HOOC-CH2 CH2-CO-COOH
(α -Ketoglutaric acid)
NH3
NH2
|
HOOC -CH2 -CH2-CH-COOH
(Glutamic acid)
NH3
NH2
|
H2N-OC-CH2-CH2-CH-COOH
(Glutamine)
HOOC CO CH2COOH
(Oxalacetic acid)
NH3
NH2
|
HOOC - CH2-CH-COOH
(Aspartic acid)
NH3 NH2
|
H2N -OC -CH2-CH-COOH
(Asparagine)
CH3 CO COOH
(Pyruvic acid)
NH3
NH2
|
CH3- CH-COOH
(Alanine)

10. Study of utilization of
radioactive isotopes in the
investigation of Biogenetic
studies.

11. Introduction
1. Living plants considered as biosynthetic laboratory primary as well
as secondary metabolite.
2. Different biosynthetic pathway:
» Shikmic acid pathway: Aromatic amino acids
» Mevalonic acid pathway: Terpenes
» Acetate pathway: Fatty acids
Biosynthesis: Formation of a chemical compound by a living
organisms
Biogenesis: Production or generation of living organisms from
other living organisms

12. Various intermediate and steps are involved in biosynthetic
pathway in plants can be investigated by means of following
techniques: -
» Use of isolated organ
» Grafting methods
» Use of mutant strain
» Tracer technique
» Enzymatic studies

13. Isolated organ, tissue & cells:
Growing of isolated organs , tissues & cells in suitable media or water is
one of the technique of investigation.
This technique is useful in the determination of site of biosynthesis of
particular compounds.
It eliminates interferences from other parts of plant which may produce
secondary change in metabolites.
ex. Roots and leaves for the study of Nicotiana and Datura, petal disc for
the study of rose oil, tropane alkaloids in the root of solanaceae family.

14. Grafting methods:
This technique uses joining two different but closely related plants together
to grow as one.
The upper portion of on plant i.e., scion grafted in stock of another plant
This method is used fore the study of alkaloid formation by grafted plants.
ex. Tomato scions grafted on Datura produce alkaloids, while Datura scion
grafted on Tomato produce less quantity of alkaloids.
This shows that main site of alkaloid biosynthesis is root.

15. Use of mutant strains:
In this mutant strains of microorganisms are produced with the
lack of certain enzymes, which results in their metabolism
being blocked at a particular stage.
ex. A mutant of Lactobacillus acidophilus, by its ability to utilize a
constituent of ‘brewer’s solubles’ but not acetate, led to the
isolation of mevalonic acid, an important intermediate of the
isoprenoid compound pathway.

16. Tracer technique:
It canbe defined as technique which utilizes a labelled compound to find
out or to trace the different intermediates and various steps in
biosynthetic pathways in plants, at a given rate & time.
OR
In this technique different isotope, mainly the radioactive isotopes which
are incorporated into presumed precursor of plant metabolites and are used
as marker in biogenic experiments.

17. The labelled compound can be prepared by use of two types of isotopes.
» Radioactive isotopes:
• [e.g. 1H, 14C, 24Na, 42K, 35S, 35P, 131I decay with emission of radiation]
• For biological investigation – carbon & hydrogen.
• For metabolic studies – S, P,and alkali and alkaline earth metals are used.
• For studies on protein, alkaloids, and amino acid – labelled nitrogen atom
give more specific information.
• 3H compound is commercially available.
» Stable isotopes:
• [e.g. 2H, 13C, 15N, 18O]
• Used for labeling compounds as possible intermediates in biosynthetic
pathways.
• Usual method of detection are: – Mass spectroscopy [15N, 18O]
• NMR spectroscopy [2H, 13C]

18. SIGNIFICANCEOF TRACERTECHNIQUE
Tracing of Biosynthetic Pathway: - e.g. By incorporation of
radioactive isotope of 14C into phenylalanine, the biosynthetic
cyanogenetic glycoside prunasin, can be detected.
Location & Quantity of compound containing tracer: - 14C labelled
glucose is used for determination of glucose in biological system
Different tracers for different studies: - For studies on nitrogen and
amino acid. (Labelled nitrogen give specific information than carbon)
Convenient and suitable technique

19. CRITERIAFOR TRACER TECHIQUE
The starting concentration of tracer must be sufficient withstand
resistance with dilution in course of metabolism.
Proper Labelling: - for proper labelling physical & chemical nature of
compound must be known.
Labelled compound should involve in the synthesis reaction.
Labelled should not damage the system to which it is used.

20. Advantages
High sensitivity.
Applicable to all living
organism.
Wide ranges of isotopes are
available.
More reliable, easily
administration & isolation
procedure.
Gives accurate result, if proper
metabolic time & technique
applied.
Disadvantages
Kinetic effect
Chemical effect
Radiation effect
Radiochemical purity
High concentration distorting
the result.

21. REQUIREMENTFORTRACER
TECHNIQUE
I. Preparation of labelled compound.
II. Introduction of labelled compound into a biological
system.
III. Separation & determination of labelled compound
in various biochemical fractions at later time.

22. I. Preparation of labelled compound.
o The labelled compound produce by growing chlorella in atmosphere of
14CO2 All carbon compounds 14C labelled.
o The 3H (tritium) labelled compound are commercially available. Tritium
labeling is effected by catalytic exchange in aqueous media by
hydrogenation of unsaturated compound with tritium gas. Tritium is pure β-
emitter of low intensity & its radiation energy is lower than 14C.
By the use of organic synthesis: -
CH3MgBr +14CO2 CH3
14COOHMgBr + H2O
CH3
14COOH + Mg(OH)Br

23. II. Introduction of labelledcompound
PRECAUTION: -
The precursor should react at necessary site of synthesis in plant.
Plant at the experiment time should synthesize the compound under investigation
The dose given is for short period.
1) Root feeding: The plant in which roots are the biosynthetic sites e.g., Tobacco.
The plants are hydroponically cultivated to avoid microbial contamination.
2) Stem feeding: Substrate can be administered through cut ends of stem
immersed in solution. For latex containing plant this method is not suitable.

24. 3)Direct injection: Suitable for plant with hollow stem e.g., umbelliferous
and opium.
4) Infiltration: Suitable for plant rooted in soil or other support without
disturbing the roots (Wick feeding).
5)Floating method: When small amount of material is available this
method is used. This technique is used in conjugation with vacuum
infiltration to remove gases.
6) Spray technique: Compound has been absorbed after being sprayed
on leaves in aqueous solution e.g., Steroids

25. III. Separation and detection of compound
Geiger – Muller counter.
Liquid Scintillation counter.
Gas ionization chamber.
Bernstein – Bellentine counter.
Mass spectroscopy.
NMR eletrodemeter.
Autoradiography.
Radio paper chromatography.

26. Geiger – Muller counter
A Geiger counter (Geiger-Muller tube) is a device used for the detection and
measurement of all types of radiation: alpha, beta and gamma radiation.
Basically it consists of a pair of electrodes surrounded by a gas. The
electrodes have a high voltage across them. The gas used is usually Helium or
Argon. When radiation enters the tube it can ionize the gas. The ions (and
electrons) are attracted to the electrodes and an electric current is produced.
A scaler counts the current pulses, and one obtains a "count" whenever
radiation ionizes the gas.

27. Liquid Scintillation counter
Scintillations literal meaning is luminescence and scintillators are material which
have property to luminate.
When ionizing particles colloids on scintillating material they absorbs its energy
and exhibits scintillation ( i.e. re-emit the absorbed energy in the form of light)
Light strikes on photosensitive surface, leads to release of photoelectron and
multiplication through the series of photo multiplier tubes(PMT).
Light converts into an electrical signal.

28. Auto-radiography
Autoradiography is the bio-analytical technique used to visualize the distribution
radioactive labelled substance in a biological sample. It is a method by which a
radioactive material can be localized within a particular tissue, cell, cell organelles
or even biomolecules.
In this method, the specimen under study is kept in contact with a suitable
photographic emulsion such as silver bromide gel or X-ray sensitive film for suitable
time period.
After the film is developed in the usual manner and results are correlated with
darkening of the film.

29. METHODS IN TRACERTECHNIQUE
1. PRECURSOR PRODUCT SEQUENCE: - In this technique, the presumed
precursor of the constituent under investigation on a labelled form is fed into the
plant and after a suitable time the constituent is isolated, purified and radioactivity is
determined.
Disadvantage: -
The radioactivity of isolated compound alone is not usually sufficient evidence that the
particular compound fed is direct precursor, because substance may enter the general
metabolic pathway and from there may become randomly distributed through a whole
range of product.
Application: -
Stopping of hordenine production in barley seedling after 15 – 20 days of
germination.
Restricted synthesis of hyoscine, distinct from hyoscyamine in Datura stramonium.
This method is applied to the biogenesis of morphine & ergot alkaloids

30. 2. DOUBLE& MULTIPLELABELLING
This method give the evidence for nature of biochemical incorporation of
precursor arises double & triple labelling. In this method specifically labelled
precursor and their subsequent degradation of recover product are more
employed. Application: -
This method is extensively applied to study the biogenesis of plant
secondary metabolite.
Used for study of morphine alkaloid. E.g. Leete, use Doubly labeled lysine
used to determine which hydrogen of lysine molecule was involved in
formation of piperidine ring of anabasine in Nicotina glauca.

31. 3. COMPETITIVE FEEDING
If incorporation is obtained it is necessary to consider whether this
infact, the normal route of synthesis in plant not the subsidiary
pathway. Competitive feeding can distinguish whether B & B’ is
normal intermediate in the formation of C from A.
Application: -
• This method is used for elucidation of biogenesis of propane alkaloids.
• Biosynthesis of hemlock alkaloids (conline, conhydrine etc) e.g. biosynthesis of
alkaloids of Conium maculactum (hemlock) using 14C labelled compounds.

32. 4. SEQUENTIAL ANALYSIS
The principle of this method of investigation is to grow plant in
atmosphere of 14CO2 & then analyze the plant at given time interval
to obtain the sequence in which various correlated compound
become labelled.
Application: -
14CO2 & sequential analysis has been very successfully used in
elucidation of carbon in photosynthesis.
Determination of sequential formation of opium hemlock and tobacco
alkaloids.
Exposure as less as 5 min. 14CO2, is used in detecting biosynthetic
sequence as –
Piperitone --------- (-) Menthone ---------- (-) Menthol in Mentha
piperita.

33. APPLICATION OFTRACER TECHNIQUE
Study of squalene cyclization by use of 14C, 3H labelled mevalonic acid.
Interrelationship among 4 – methyl sterols & 4, 4 dimethyl sterols, by use of 14C
acetate.
Terpenoid biosynthesis by chloroplast isolated in organic solvent, by use of 2- 14C
mevalonate.
Study the formation of cinnamic acid in pathway of coumarin from labelled coumarin.
Origin of carbon & nitrogen atoms of purine ring system by use of 14C or 15N labelled
precursor.
Study of formation of scopoletin by use of labelled phenylalanine.
By use of 45Ca as tracer, - found that the uptake of calcium by plants from the soil.
(CaO & CaCO2).
By adding ammonium phosphate labelled with 32P of known specific activity the
uptake of phosphorus is followed by measuring the radioactivity as label reaches
first in lower part of plant, than the upper part i.e. branches, leaves etc.

34. THANK YOU !!!
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